Lactobacillus transformation (Speer 2012)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(→Procedure)
Line 12: Line 12:
*0.5M Sucrose, 10% glycerol solution
*0.5M Sucrose, 10% glycerol solution
-
==Procedure==
+
°C==Procedure==
-
#Grow L. plantarum overnight from freezer stock in 5ml MRS medium (30°C without shaking).
+
#'''Grow ''L. plantarum'''''
-
#Add 1g glycine to two flasks of 50ml MRS medium to make 100ml 2% glycine.
+
##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.
 +
##Grow overnight at 30°C without shaking.
 +
#'''Make 100ml 2% glycine:'''
 +
##Add 1g glycine to two flasks of 50ml MRS medium.
##Shake to dissolve.
##Shake to dissolve.
-
#Add 1ml overnight culture to each flask (1:50 dlution)
+
#'''Add 1ml overnight culture to each flask (1:50 dlution).'''
-
#Culture for ~3 hours.
+
#'''Culture for ~3 hours at 37°C with shaking.'''
 +
#'''Wash 2x with ice-cold DI water:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold DI water.
 +
##Repeat.
 +
#'''Treat with ice-cold EDTA:'''
 +
##Centrifuge for 5min at 4000g
 +
##Resuspend in 10ml 50mM EDTA.
 +
##Incubate on ice for 5 minutes
 +
#'''Wash with ice-cold DI water:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold DI water.
 +
#'''Wash 2x with Electroporation Buffer:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
 +
##Repeat.
 +
#'''Concentrate cells:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
 +
#'''Divide cells:'''
 +
##Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
 +
##Keep on ice until use (within the next two hours).
 +
#'''Add DNA:'''
 +
##Add 10μL of plasmid DNA to the 90μL of cell concentrate.
 +
##Keep on ice for 5 minutes.
 +
##Put 1mm cuvettes on ice too.
 +
#Electroporate:
 +
##Pipette the cell/DNA mixture into the cuvette
 +
##Electrporate at 1200 volts
 +
##Time constant should be ~5.0
 +
#Recovery:
 +
##Immediately transfer the electroporated cells to 900μL of MRS medium.
 +
##Incubate at 30°C for 2-3 hours.
 +
#Select:
 +
##Plate on medium with the appropriate antibiotic
 +
##Incubate at 30°C for two days.
 +
##Pick colony
==Notes==
==Notes==

Revision as of 15:59, 14 September 2011

Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Back to Richard Lab:protocols

Contents

Overview

This is basically a modified version of the Mason (2005 protocol) for use with Lactobacillus plantarum and an Eppendorf 2510 electroporator.

Materials

  • MRS media
  • Glycine
  • Milipore water
  • 50mM EDTA solution
  • 0.5M Sucrose, 10% glycerol solution

°C==Procedure==

  1. Grow L. plantarum
    1. Inoculate 5ml MRS medium with L. plantarum freezer stock.
    2. Grow overnight at 30°C without shaking.
  2. Make 100ml 2% glycine:
    1. Add 1g glycine to two flasks of 50ml MRS medium.
    2. Shake to dissolve.
  3. Add 1ml overnight culture to each flask (1:50 dlution).
  4. Culture for ~3 hours at 37°C with shaking.
  5. Wash 2x with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
    3. Repeat.
  6. Treat with ice-cold EDTA:
    1. Centrifuge for 5min at 4000g
    2. Resuspend in 10ml 50mM EDTA.
    3. Incubate on ice for 5 minutes
  7. Wash with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
  8. Wash 2x with Electroporation Buffer:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
    3. Repeat.
  9. Concentrate cells:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  10. Divide cells:
    1. Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
    2. Keep on ice until use (within the next two hours).
  11. Add DNA:
    1. Add 10μL of plasmid DNA to the 90μL of cell concentrate.
    2. Keep on ice for 5 minutes.
    3. Put 1mm cuvettes on ice too.
  12. Electroporate:
    1. Pipette the cell/DNA mixture into the cuvette
    2. Electrporate at 1200 volts
    3. Time constant should be ~5.0
  13. Recovery:
    1. Immediately transfer the electroporated cells to 900μL of MRS medium.
    2. Incubate at 30°C for 2-3 hours.
  14. Select:
    1. Plate on medium with the appropriate antibiotic
    2. Incubate at 30°C for two days.
    3. Pick colony

Notes

All questions, input and feedback are welcome!

References

There will be one here soon.


Contact

  • mas853@psu.edu

or instead, discuss this protocol. -->

Retrieved from "http://openwetware.org/wiki/Lactobacillus_transformation_%28Speer_2012%29"
Personal tools